Hedgehog (Hh) signaling coordinates events in embryogenesis but is largely quiescent in differentiated tissue. Uncontrolled activation of the pathway is implicated in a variety of human cancers, including pediatric medulloblastoma and essentially all basal cell carcinomas. Its causative role in oncogenesis highlights this pathway as a therapeutic target, and the first FDA-approved Hh antagonist (vismodegib) has shown remarkable tumor inhibition in advanced basal cell carcinomas. Despite its initial success, cases of vismodegib resistance have begun to emerge, and developmental defects in mice caused by analogs raises concerns about its use in children. Next-generation therapies are necessary to overcome these limitations and fully realize the clinical potential of Hh pathway inhibition. Normal signaling is stimulated when the Hh ligand binds to the receptor Patched (Ptc), which releases inhibition of the GPCR-like protein Smoothened (Smo). Smo inhibits the negative pathway regulator Suppressor of Fused (Sufu) and effects activation of the Gli transcription factors (Gli1, Gli2, and Gli3) that mediate pathway activity. Since vismodegib and nearly all small molecule Hh inhibitors antagonize Smo, cancers initiated downstream of Smo are insensitive to these compounds. Smo is also particularly susceptible to mutations that confer resistance after sustained treatment. To find a pathway antagonist that circumvents these limitations, the Chen lab recently completed a screen of 325,120 compounds for their ability to inhibit pathway activity induced by loss of Sufu. These studies identified an imidazolium compound, henceforth termed glimidazole, that selectively blocks Gli1 activity but leaves Gli2 and Gli3 intact. Due to its direct inhibition of the Gli1, glimidazole compounds have potential as a broad-spectrum treatments for Hh-dependent cancers. Furthermore, while Gli2 and Gl3 are essential for development, Gli1-/- mice show no obvious defects, suggesting that Gli1-selective antagonists may provide a therapy for childhood patients. To develop the clinical potential of glimidazole derivatives, we will (1) identify and functionally characterize the cellular target of glimidazole, and (2) assess the effects of glimidazole analogs on tumorigenesis and murine development. We will synthesize glimidazole derivatives to enable the isolation of specific binding proteins and characterize interactors by mass spectrometry-based sequencing. Concurrently, we will use whole-transcriptome comparisons of glimidazole-sensitive and glimidazole-resistant cells to identify potential glimidazole targets. To discern the physiologicaly relevant glimidazole target from other interacting proteins, we will assess their roles in Hh pathway regulation through loss- and gain-of-function perturbations. We will exploit a modular chemical synthesis of glimidazole structures to optimize their potency, selectivity, and pharmacokinetic properties through medicinal chemistry. In collaboration with Prof. Jean Tang, we will evaluate the most promising analogs in a basal cell carcinoma allograft model, and compounds that inhibit tumor progression will be evaluated in juvenile mice for teratogenic effects.